Identification and Characterization of PSEUDO-RESPONSE REGULATOR (PRR) 1a and 1b Genes by CRISPR/Cas9-Targeted Mutagenesis in Chinese Cabbage (Brassica rapa L.)

Abstract

The CRISPR/Cas9 site-directed gene-editing system offers great advantages for identifying gene function and crop improvement. The circadian clock measures and conveys day length information to control rhythmic hypocotyl growth in photoperiodic conditions, to achieve optimal fitness, but operates through largely unknown mechanisms. Here, we generated core circadian clock evening components, Brassica rapa PSEUDO-RESPONSE REGULATOR (BrPRR) 1a, 1b, and 1ab (both 1a and 1b double knockout) mutants, using CRISPR/Cas9 genome editing in Chinese cabbage, where 9–16 genetic edited lines of each mutant were obtained. The targeted deep sequencing showed that each mutant had 2–4 different mutation types at the target sites in the BrPRR1a and BrPRR1b genes. To identify the functions of BrPRR1a and 1b genes, hypocotyl length, and mRNA and protein levels of core circadian clock morning components, BrCCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) and BrLHY (LATE ELONGATED HYPOCOTYL) a and b were examined under light/dark cycles and continuous light conditions. The BrPRR1a and 1ab double mutants showed longer hypocotyls, lower core circadian clock morning component mRNA and protein levels, and a shorter circadian rhythm than wildtype (WT). On the other hand, the BrPRR1b mutant was not significantly different from WT. These results suggested that two paralogous genes may not be associated with the same regulatory function in Chinese cabbage. Taken together, our results demonstrated that CRISPR/Cas9 is an efficient tool for achieving targeted genome modifications and elucidating the biological functions of circadian clock genes in B. rapa, for both breeding and improvement.

Transcriptome Analysis of Intermittent Light Induced Early Bolting in Flowering Chinese Cabbage

“…There have been experiments proving that LHY overexpression in plants results in a late-flowering phenotype [ 56 ]. The flowering time of lhy in long-day conditions is the same as the wild-type, but it advances in short-day environments [ 57 ]. Our research results show that BrLHY in the treatments decreases during stage S2, while there is little change in the CK.…”

Dissection of the E8 locus in two early maturing Canadian soybean populations

“…(2021) investigated the role of Glyma.04G167900 ( GmLHCA4 ) and observed a 1.8-day difference in the number of days to flowering between two GmLHCA4 haplotypes. The PSEUDO RESPONSE REGULATOR (PRR) family regulates many biological processes in Arabidopsis, including photoperiodic flowering, growth, stress response, and regulation of the circadian clock ( Hayama et al., 2017 ; Li et al., 2019 ; Kim et al., 2022 ), with several homologs found within the soybean genome. The domestication of the Glyma.12G073900 ( GmPRR3b ) gene in soybean has been associated with an early flowering phenotype due to the presence of a causal SNP at position GM12:5,520,945 ( Li et al., 2020 ).…”

The Roles of Circadian Clock Genes in Plant Temperature Stress Responses

“…Additionally, considering that clock genes are involved in responses to various environmental stresses such as salt and drought, studies of clock genes and expression regulation technologies are important for efforts to improve plant characteristics. Various efforts have been made to mutate clock genes to improve various crop characteristics, including RNAi and overexpression, as well as recent gene-editing techniques (Table 1) [89,90]. In recent years, global warming has emerged as an important ecological problem for vegetation and crop agriculture worldwide [86]; thus, further analyses of the relationships between temperature stress response and plant growth and development are necessary to secure future plant resources and food security.…”